Troponin i-induced cardiac inflammation and dysfunction in mice: a comparative study with the AT-3 tumor-bearing model

Abstract

Background

Myocarditis is a potentially fatal condition, with a mortality rate of up to 50% in severe cases. Studies, including those by Nobel Laureate Honjo, have implicated autoantibodies against cardiac troponin I (cTnI) in driving cardiac inflammation in mice. Research has also identified autoantibodies under baseline conditions in some cancer models. However, data on the effects of recombinant cTnI on autoantibody production, myocardial inflammation, and contractile function remain limited. This study investigated cTnI-associated myocardial inflammation and autoantibody formation in both tumor-free and tumor-bearing mouse models.

Methods

Female BALB/c mice were immunized with recombinant cTnI combined with adjuvants and compared to adjuvant-only controls. Cardiac function was assessed using gated cardiac MRI, including myocardial velocities, acceleration, deceleration, and standard volumetric parameters including ejection fraction (EF). Anti-cTnI autoantibodies were quantified using a custom-designed ELISA, while myocardial inflammation was assessed by analyzing T-cell subsets (CD4 + and CD8 +) in myocardial tissue samples. Baseline autoantibody reactivity was evaluated in tumor-bearing mice and tumor-free controls for comparison.

Results

The left ventricular ejection fraction trended lower in the cTnI + adjuvant group (57.80 ± 1.7%) compared to controls (61.67 ± 4.1%), but the difference was not statistically significant (p = 0.073). Myocardial velocity, reflecting contraction speed, was significantly reduced in cTnI-treated mice (control:-1.2 ± 0.8 cm/s; cTnI:-1.05 ± 0.07 cm/s; p = 0.015). Anti-cTnI autoantibody levels increased significantly in cTnI-treated mice at 8 weeks (control:0.1 ± 0.02; cTnI:0.77 ± 0.28; p = 0.007). Additionally, the density of CD8 + T-cells in myocardial tissue was significantly higher in the cTnI group (control:2.2 ± 1.2 cells/mm²; cTnI:4.4 ± 2 cells/mm²; p = 0.013), indicating an enhanced cytotoxic T-cell response. The CD4/CD8 ratio was significantly lower in cTnI-treated mice (control: 8.2 ± 6.8; cTnI:3.1 ± 0.9; p = 0.029), further suggesting a shift toward a cytotoxic immune profile. Baseline autoantibody reactivity in tumor-bearing mice was not significantly different from controls (tumor-bearing: absorbance 0.049 ± 0.029; control: absorbance 0.068 ± 0.05 at 450 nm), indicating no inherent autoimmune reactivity in the tumor-bearing model.

Conclusions

Recombinant cTnI induces myocardial contractile dysfunction and promotes a cytotoxic immune response, supporting its role as an autoantigen in myocarditis. Advanced cardiac MRI revealed subtle functional impairments that EF alone could not detect. These findings highlight the potential for therapies targeting cTnI-induced autoimmunity, particularly in patients with ICI-associated myocarditis.

Introduction

Myocarditis is an inflammatory condition with various causes, including infections, autoimmune responses, and adverse effects from cancer therapies, particularly immune checkpoint inhibitors (ICIs) [1,2,3,4]. In patients treated with cancer therapies, myocarditis can emerge as a serious, life-threatening condition linked to immune responses against cardiac tissue [2, 5]. While T-cell involvement in myocarditis is well-established, B cells and autoantibodies are increasingly recognized as significant contributors to its pathology [6, 7]. Autoantibodies against cardiac proteins like cardiac troponin I (cTnI) have been implicated in intensifying myocardial damage [6,7,8]. Current treatments, which often involve administering high-dose steroids and intravenous immunoglobulin (IVIG), are limited by the risk of immune suppression or cancer progression [9, 10]. This limitation highlights the need for specific therapies that target autoimmune mechanisms in myocarditis without impairing cancer treatment.

Previous studies by Kaya et al. and Goser et al. demonstrated that cardiac troponin I (cTnI) induces myocardial inflammation and fibrosis but did not examine its effects on myocardial contractile function or immune cell dynamics [11, 12]. They also did not evaluate baseline autoantibody reactivity or the effects of cTnI in tumor-bearing models. Our study addresses these gaps by investigating the functional effects of cTnI-driven immune reaction, analyzing immune cell alterations, and examining autoantibody production in both tumor-free and tumor-bearing mice. Furthermore, no studies have directly assessed cTnI-induced myocarditis using cardiac magnetic resonance imaging (MRI), an advanced tool that provides detailed information on myocardial function, including myocardial strain and velocity changes.

Unlike traditional echocardiography, cardiac MRI offers comprehensive assessments of myocardial inflammation and function and allows for the detection of subtle changes that may precede overt dysfunction [3, 13, 14]. These imaging parameters are particularly useful in autoimmune myocarditis, where early inflammatory changes may not be evident with standard measures such as ejection fraction or wall motion. Cardiac MRI thus provides a critical tool for evaluating the effects of cTnI on myocardial injury and may help identify early signs of subtle or sub-clinical cardiac dysfunction.

The rationale for this study stems from the current limited understanding of how full-length cTnI contributes to myocardial inflammation and autoantibody production, despite its suggested role as a key autoantigen in myocarditis. Furthermore, the presence of baseline autoantibodies in cancer models on cTnI-driven immune responses remains unexplored. Therefore, we administered recombinant cTnI protein to murine models to directly study its effects in inducing autoantibodies and myocardial inflammation. We characterized the inflammatory response by analyzing cardiac tissue for CD4 + and CD8 + T cells and measured circulating autoantibodies against cTnI to explore the connection between immune sensitization and autoantibody production. Using tumor-bearing controls allowed us to establish baseline autoantibody levels in the presence of cancer.

Methods

The animal care and experimental protocols followed US National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of the Roswell Park Comprehensive Cancer Center (RPCCC). Experiments used 5-week-old female BALB/c mice (Charles River Laboratories).

Immunization of a mouse model with recombinant cardiac troponin I protein

Two immunization protocols were implemented in this study to evaluate the immunogenicity of recombinant cardiac troponin I (cTnI) protein: a low-dose protocol and a high-dose protocol, each with specific adjuvant regimens (Fig. 1).

Fig. 1

Timeline and dosing regimen of low- and high-dose cTnI immunization protocols with adjuvants in a mouse model

In the low-dose protocol, mice were immunized intramuscularly with 2.5 µg of recombinant cTnI protein suspended in phosphate-buffered saline (PBS) and mixed with aluminum hydroxide gel (alhydrogel) adjuvant. Injections were administered at weeks 0 followed by booster dose at week 3 to provide a priming of the immune response.

The high-dose protocol involved initial 3 doses of 100 ng cTnI with alhydrogel 3 weeks apart (week 0, week 3 and week 6) followed by additional booster phase with Freund’s adjuvants to enhance the immune response. At week 9, mice were injected intramuscularly with 45 µg of cTnI protein mixed with 310 µL of Complete Freund’s Adjuvant (CFA). This was followed by two additional booster injections at weeks 10 and 11, each consisting of 45 µg of cTnI protein combined with 190 µL of Incomplete Freund’s Adjuvant (IFA).

Control groups received the same injections with PBS or adjuvants without cTnI protein to serve as controls for vehicle and adjuvant effects.

Blood samples were collected retroorbitally under isoflurane anesthesia to assess early immune responses. Final blood samples were obtained via cardiac puncture during termination by CO₂ euthanasia at week 10 -12. Key analyses included cardiac MRI to detect structural changes, ELISA to measure anti-cTnI autoantibody levels, and immunohistochemical analysis to evaluate histological changes in cardiac tissues.

The sample size ranged from 5 to 10 mice per cohort in the cTnI injection groups and from 3 to 8 mice per cohort in the control groups. This dual-phase protocol, combining low-dose priming with alhydrogel and high-dose boosting with Freund’s adjuvants, was designed for a clear assessment of cTnI-induced immune and cardiac responses.

Cardiac magnetic resonance imaging to study left ventricular contractility and volumes

Prior to the termination of the experiment, magnetic resonance imaging (MRI) was conducted using a 4.7 Tesla preclinical scanner with a 35-mm internal diameter, quadrature transceiver coil and ParaVision 4.0 acquisition platform (Bruker Biospin, Billerica, MA), as described previously by our group [15]. During imaging, mice were anesthetized and maintained with 2–2.5% isoflurane. Body temperature, heart rate, and respiration were monitored and maintained with compatible gating, as formerly described [15]. Analysis of left ventricular (LV) volumes and function and phase-specific (both systolic and diastolic velocity mapping) was quantified using the software Segment version 2.2 R6901, also previously described by our group [13].

Tissue immunohistochemistry to detect myocardial inflammation

Formalin-fixed, 4-μm-thick paraffin-embedded sections were placed on charged slides and dried at 60 °C for one hour (Pathology Network Shared Resource, Roswell Park). Slides were cooled to room temperature and added to the Dako Omnis autostainer, where the sections were deparaffinized and rehydrated. Sections were incubated for 30 min in Flex TRS high pH (Dako, GV80411-2) for target retrieval. Slides were then incubated with anti-CD4 (Abcam ab183685) and anti-CD8 (Abcam ab209775) for 30 min. Rabbit Envision (Agilent K4003) was used for 30 min and then with a Dab chromogen for 5 min for the visualization of T-cells. Slides were counterstained with hematoxylin for 8 min. Sections were scanned using Aperio Scanner (at 40 × magnification) and positively stained cells were counted in the whole slide using a counter pen tool in Aperio ImageScope.

Baseline anti-troponin I autoantibody development in tumor-bearing and non-tumor-bearing mice

Anti-troponin I autoantibody levels were assessed in both tumor-bearing and non-tumor-bearing mice. For the tumor-bearing group, a syngeneic mouse tumor model was created by injecting 200,000 AT-3 mouse mammary carcinoma cells subcutaneously into the mammary fat pad of C57BL/6 mice. Mice were monitored closely for tumor progression and euthanized between weeks four and five when tumors began to ulcerate. For the non-tumor-bearing group, age-matched control mice were used without any tumor injection.

Serum samples from both groups were analyzed for the presence of anti-troponin I antibodies. To detect cTnI-reactive antibodies, 0.05 µg of protein (LifeDiagnostics, 7110 for mouse cTnI) was coated per well in Nunc MaxiSorp plates (Thermofisher, 44–2404-21) overnight. Mouse serum samples were diluted 1:30 in 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) and incubated overnight at 4 °C. Goat anti-mouse IgGH + L (115–035-003; Jackson ImmunoResearch) was diluted 1:10,000 in 2% BSA in PBS and applied as the secondary antibody. After washing, TMB (3,3’,5,5’-tetramethylbenzidine) substrate solution (34,021; Thermo Fisher Scientific) was added, and the reaction was stopped with 2 M sulfuric acid. The absorbance at 450 nm (A450) was measured to quantify antibody levels.

Statistical analyses

Descriptive statistics were presented as means with standard deviations (SDs) for continuous variables. For comparisons between two groups, unpaired t-tests were applied to endpoints including myocardial velocity, acceleration, deceleration, and anti-cTnI antibody levels. Statistical significance was determined using a two-sided p value < 0.05. All analyses were performed using GraphPad Prism version 10.4.0.

Results

Cardiac MRI-detected subtle cardiac dysfunction in mice following cTnI + adjuvant treatment

Recombinant cardiac troponin I (cTnI) administration led to impaired cardiac function, with MRI metrics showing significant changes. Left ventricular ejection fraction (LVEF) trended lower in the cTnI + adjuvant group (57.8 ± 1.8%) compared to controls (61.7 ± 4.1%) but was not statistically significant (p = 0.073) (Fig. 2A). Velocity, representing myocardial contraction speed, was significantly reduced in treated mice (cm/s: control, -1.2 ± 0.08, cTnI, -1.05 ± 0.06, p = 0.015) (Fig. 2B). Both peak systolic acceleration (cm/s²: control, -9.7 ± 1, cTnI, -8.1 ± 0.5, and deceleration (cm/s²: control, 9.4 ± 0.6, cTnI, 8.0 ± 0.5), indicators of the heart’s ability to rapidly contract and relax, were also significantly impaired in the treatment group (p = 0.019 and p = 0.016, respectively) (Fig. 2C/D). These sensitive MRI measures reveal reduced contractile and relaxation efficiency following cTnI + adjuvant exposure, which indicated subtle but measurable cardiac dysfunction.

Fig. 2

Comparison of cardiac function in control versus cTnI (experimental) groups using gated cardiac MRI. A Ejection fraction showed a trend toward reduction in the experimental group, but this difference was not statistically significant. B Myocardial velocity was significantly lower in the cTnI compared to controls (p = 0.0146). C, D Both acceleration and deceleration rates were significantly reduced in the experimental group, with p-values of 0.0198 and 0.0160, respectively. Bars show mean ± SD, with individual data points displayed as dot plots

Time-dependent increase in anti-cTnI autoantibodies following cTnI + adjuvant treatment

Anti-cTnI autoantibody production was assessed in cTnI + adjuvant-treated mice compared to controls. After 3 weeks, treated mice showed a 2.2-fold increase in serum reactivity against cTnI protein (0.22 ± 0.08 absorbance unit, AU) compared to controls (0.10 ± 0.08 AU), though this difference was not statistically significant (p = 0.09) (Fig. 3A). At 8 weeks, there was a substantial 7.8-fold increase in anti-cTnI antibody levels in treated mice, reaching statistical significance (control, 0.1 ± 0.02 AU, cTnI, 0.78 ± 0.28 AU, p = 0.007) (Fig. 3B). This indicates a progressive and significant elevation in autoantibody production against cTnI over time in response to the cTnI + adjuvant treatment.

Fig. 3

ELISA analysis of serum antibody reactivity against cardiac troponin I (cTnI) in control or cTnI injected mice, measured at 3 and 8- weeks post-treatment. Absorbance values at 450 nm reflect anti-cTnI antibody levels. Comparisons between control and cTnI-treated groups at both 3 weeks (panel A) and 8 weeks (panel B) indicate increased antibody reactivity in cTnI-treated mice, with statistical significance at 8 weeks. Bars show mean ± SD, with individual data points displayed

Increased cytotoxic T-cell response in cTnI-treated myocardial tissue

In this experiment comparing control and cTnI-treated groups (with adjuvant) in myocardial tissue, CD4, CD8, and the CD4/CD8 ratio were analyzed. CD4 density (CD4/mm²) showed no significant difference between groups (control, 13.3 ± 6.1, cTnI, 13.6 ± 5.4, p = 0.921) (Fig. 4A), which represents similar levels of helper T-cells. CD8 density (CD8/mm²) was significantly elevated in the cTnI-treated group (control, 2.2 ± 1.2, cTnI, 4.4 ± 2, p = 0.013) (Fig. 4B), which suggests an enhanced cytotoxic T-cell response. The CD4/CD8 ratio was significantly lower in the cTnI-treated group (control, 8.2 ± 6.8, cTnI, 3.1 ± 0.9, p = 0.029), indicating a shift toward a cytotoxic profile (Fig. 4C). These findings suggest that cTnI treatment may promote a cytotoxic-dominant immune response in myocardial tissue.

Fig. 4

Immunohistochemistry of CD4, CD8 Levels, and CD4/CD8 Ratio in Mouse Myocardial Specimens (Control vs. cTnI-Treated). Immunohistochemistry of mouse myocardial specimens shows CD4, CD8, and the CD4/CD8 ratio in control (blue) and cTnI-treated (red) groups. Panel A CD4 levels remain unchanged between groups. Panel B CD8 levels are significantly higher in the cTnI-treated group, confirming an increase in cytotoxic T-cell activity. Panel C The CD4/CD8 ratio is significantly lower in the cTnI-treated group, indicating a shift toward a cytotoxic profile. Bars show mean ± SD, with individual data points displayed as dot plots

Absence of baseline autoimmune reactivity in mammary tumor-bearing mice

In this study, mice injected with AT-3 mammary tumor cells developed tumors within one week, with measurable growth by the second week. Control mice without tumors exhibited anti-cTnI antibody levels with an absorbance of 0.068 ± 0.05 at 450 nm, while tumor-bearing mice showed lower levels, with an absorbance of 0.049 ± 0.029 at 450 nm. No significant difference in antibody levels was observed between the groups, indicating no baseline autoimmune reactivity in mice with mammary tumors (Fig. 5).

Fig. 5

Absorbance values at 450 nm for anti-cTnI antibodies in non-tumor (control) and AT-3 tumor-bearing mice. No significant difference in anti-cTnI levels was observed between the groups (p = 0.5101). Bars show mean ± SD, with individual data points displayed

Discussion

In this study, we demonstrate that cardiac troponin I (cTnI) can serve as a potential immunological target in myocarditis, evidenced by the induction of an inflammatory response with myocardial inflammation CD8 + T-lymphocyte infiltration following its administration in mice. cTnI is a structurally complex protein with highly conserved motifs, such as the I-T arm and the C-terminal domain, which are involved in complex interactions within the contractile apparatus of cardiac muscle [16, 17]. Its exposed regions, coupled with a series of phosphorylation sites [18], contribute to its potential immunogenicity, making it a feasible target for autoimmune reactions [17, 19]. Previous in silico studies have reported that specific domains within cTnI may contain epitopes capable of eliciting T-cell and B-cell responses [20, 21]. Those reports reinforced the potential of cTnI to drive immune activation. While this study did not explicitly dissect the mechanistic pathways involved, these findings point to the likelihood that cTnI could play a role in provoking the observed immune responses Fig. 6.

Fig. 6

Central Illustration. Cardiac troponin I (cTnI) immunization in mice leads to elevated anti-cTnI autoantibody levels, myocardial inflammation with CD8+ T-cell infiltration, and a reduced CD4/CD8 ratio. Cardiac MRI shows impaired myocardial contractile function, including reduced velocity, acceleration, and deceleration, suggesting autoimmune process and implications for immune checkpoint inhibitor therapy.

Traditional cardiac imaging tools like echocardiography provide limited information on the inflammatory and functional changes typical in myocarditis, as they primarily measure broad metrics like ejection fraction, chamber size, and general wall motion. Cardiac MRI, especially high-field imaging, offers a more comprehensive examination, allowing for specific assessments of myocardial contractile velocities, and both systolic and diastolic functions via acceleration and deceleration measures [3, 13, 22]. These MRI-based metrics deliver a clearer picture of myocardial function, particularly in cases of myocarditis where immune-mediated damage may be subtle and not easily detectable through traditional imaging approaches [23, 24]. However, cardiac MRI has its limitations, such as high cost, limited accessibility, and the requirement for high-field technology, which may restrict its use, especially in settings where advanced imaging is unavailable.

Our approach of administering cTnI with an aluminum adjuvant was designed to stimulate an autoimmune response mimicking myocarditis. Findings of reduced peak systolic acceleration suggest that early systolic contraction was impaired, likely due to inflammation-induced myocardial stiffness, which compromises the ability of the myocardium to contract with the force seen in healthy tissue. Furthermore, reduced peak systolic deceleration indicates compromised myocardial relaxation, potentially due to a disruption in filling dynamics. This reduced contractility and relaxation likely leads to suboptimal cardiac performance, as reflected in lower LVEF. The inflammatory response within the myocardium may also disrupt structural integrity, contributing to functional decline.

While cTnI has been implicated as a target antigen in myocarditis associated with ICI therapy [7, 11], this study’s approach broadens the application by examining myocarditis in a non-cancer context, which has implications beyond cancer therapy. Identifying cTnI as a driver of autoimmune myocarditis provides a platform to develop therapies that specifically target autoimmunity without interrupting cancer treatment. This approach is particularly relevant in ICI-induced myocarditis, where discontinuing life-saving cancer therapy may lead to tumor progression. The increase in CD8 levels and reduction in the CD4/CD8 ratio in cTnI-treated myocardial tissue points to a dominant cytotoxic T-cell response [25]. This shift likely promotes direct cell damage, as CD8 T-cells are associated with targeted cytotoxicity. The lower CD4/CD8 ratio suggests limited helper T-cell activity, which may reduce regulatory functions and favor prolonged inflammation [26], potentially leading to myocardial injury or fibrosis. Future studies should address the role of B cells and other immune mediators in cTnI-induced myocarditis, as recent models suggest B-cell infiltration in myocardium also contributes to the severity of inflammation and immune activation.

Limitations

This study has certain limitations. It was a pilot study with a small sample size, particularly for cardiac MRI, due to technical constraints and the challenges of imaging at high heart rates. The AT-3 tumor model was used solely to assess baseline antibody production, as the high morbidity and rapid progression of the AT-3 tumors prevented cardiac MRIs in these mice. While data trends were clear, the study would benefit from larger sample sizes and additional models to confirm findings and expand their generalizability. However, these initial findings provide promising data trends and novel insights into cTnI-mediated autoimmune response and cardiac dysfunction. Another limitation of our study is that while trends towards lower LVEF were observed in the cTnI group, these differences did not reach statistical significance, potentially limiting the ability to draw definitive conclusions about global myocardial dysfunction. The reliance on cardiac MRI for detecting subtle changes highlights the challenge of identifying early myocardial dysfunction, which may not yet manifest as significant alterations in standard functional parameters like LVEF.

Conclusions and future directions

Our findings suggest that cTnI has the potential to induce cardiac inflammation and dysfunction, supporting its role as a key autoantigen in myocarditis. Future research should focus on identifying specific cTnI epitopes with immunogenic potential that could serve as therapeutic targets. Developing strategies to modulate immune responses to cTnI without impairing cancer treatment may provide a therapeutic path forward for patients on ICI therapy who develop myocarditis. Additionally, therapies targeting cTnI autoimmunity could have broader implications, benefiting patients with other forms of autoimmune myocarditis.